Eukaryotic cells insure that the two daughter cells from a mitotic division each receive an identical complement of DNA by the precise replication and segregation of chromosomes. The study of molecular mechanism of chromosome segregation is justified because this process is essential for the proper transmission of genetic material in all dividing cells, and a failure to execute it properly leads to cell death or aneuploidy which causes birth defects and probably contributes to formation of tumors. However, most questions about the mechanism of chromosome segregation remain unanswered. For example, how do chromosomes attach to the microtubules of the spindle, the macromolecular machine that mediates chromosome segregation? Which molecules generate chromosome movement and what is their mode of action? In this proposal, the molecular mechanism of chromosome segregation is analyzed in the yeast, Saccharomyces cerevisiae, which appears to be an excellent model organism for understanding basic cellular processes like chromosome segregation that are common to all eukaryotes from yeast to man. The proposal focuses on the centromere, the organelle of a chromosome that mediates its attachment to the spindle by binding microtubules and that generates some of the force that moves chromosomes apart during the segregation of chromosomes to the poles of the spindle. In this proposal, a novel in vitro assay is described for measuring the first of these reactions, the binding of centromeres to microtubules. This assay has been used to detect microtubule binding activity for centromeres isolated from cells as well as for those assembled in vitro by mixing extracts of yeast cells with naked centromere DNA. This assay will be used to determine the in vitro affinity of wild-type centromeres for microtubules, the size of the pool of free centromere factors not bound to CEN DNA, and the in vitro rate of assembly and disassembly of wild-type centromere DNA with the centromere factors necessary for microtubule binding. It will also be used to determine the position on the microtubule where centromeres are bound and the number of microtubules that an individual centromere can bind. The characterization of wild-type centromeres in vitro will provide a basis for using this assay in combination with genetic and cytological methods to dissect the in vivo function of the different structural elements of the centromere DNA, to address whether the assembly of yeast centromeres or their ability to bind microtubules is regulated during the cell cycle, to identify and purify the factors that bind to the centromere DNA with the aim of reconstituting the microtubule binding activity of the centromere from pure components, and to identify amino acids of tubulin that are important for controlling the affinity of yeast centromeres for microtubules in order to elucidate how centromeres and microtubules physically interact.